ABSTRACT

Centrosome cohesion, mostly regarded as a proteinaceous linker between parental centrioles, ensures that the interphase centrosome(s) function as a single microtubule-organizing center. Impairment of centrosome cohesion leads to the splitting of centrosomes. Although the list of cohesion proteins is growing, the precise composition and regulation of centrosome cohesion are still largely unknown. In this study, we show that the centriolar protein centlein (also known as CNTLN) localizes to the proximal ends of the centrioles and directly interacts with both C-Nap1 (also known as Cep250) and Cep68. Moreover, centlein complexes with C-Nap1 and Cep68 at the proximal ends of centrioles during interphase and functions as a molecular link between C-Nap1 and Cep68. Depletion of centlein impairs recruitment of Cep68 to the centrosomes and, in turn, results in centrosome splitting. Both centlein and Cep68 are novel Nek2A substrates. Collectively, our data demonstrate that centrosome cohesion is maintained by the newly identified complex of C-Nap1–centlein–Cep68.

INTRODUCTION

The mammalian centrosome is the primary microtubule-organizing center (MTOC) and comprises two centrioles and the surrounding pericentriolar material (PCM) (Nigg and Raff, 2009). The two centrioles are structurally and functionally distinct. Only the mother centriole, harboring distal and subdistal appendages, can migrate to the cell cortex to become a basal body for the formation of cilia during cell cycle quiescence (Kobayashi and Dynlacht, 2011). The ultrastructure shows that the centriole is polarized along its long axis, with the base referred to as the proximal end and the tip as the distal end (Gönczy, 2012). The two centrioles within a G1 centrosome are connected through a proteinous linker, which is often referred to as centrosome cohesion (Barrera et al., 2010) or the ‘G1–G2 tether’ (Nigg and Stearns, 2011), emanating from their proximal ends. As cells progress from G1 into the G2 phase, the centrosome is duplicated, and the duplicated centrosomes remain linked in order to function as a single MTOC (Nigg and Stearns, 2011). At the onset of mitosis, centrosome cohesion is disassembled by Nek2A-kinase-mediated phosphorylation, concomitant with centrosome separation in preparation for mitotic spindle assembly (Faragher and Fry, 2003). Proteins implicated in the tethering of the parental centrioles include C-Nap1 (also known as Cep250) (Mayor et al., 2000), rootletin (Bahe et al., 2005), Cep68 (Graser et al., 2007a), LRRC45 (He et al., 2013) and CDK5RAP2 (Graser et al., 2007a; Barrera et al., 2010), of which C-Nap1, rootletin and LRRC45 are phosphorylated by Nek2A and displaced from the centrosomes at the onset of mitosis. Despite a growing number of tethering proteins that have been identified, the precise composition of centrosome cohesion remains to be elucidated. Here, we characterize the centriolar protein centlein (also known as CNTLN) and demonstrate that centlein is complexed with C-Nap1 and Cep68, and is required for centrosome cohesion.

RESULTS AND DISCUSSION

Centlein localizes to the proximal ends of centrioles

To characterize centlein, we generated both mouse and rat monoclonal antibodies spanning residues 89–437 and 901–1191, respectively, of the recombinant centlein protein (‘antigen’, supplementary material Fig. S1A). Western blotting showed that the rat antibody clone 7H2 recognized a band of ∼170 kDa, which was greatly diminished after siRNA-mediated depletion of centlein in U2OS and RPE-1 cell lysates (supplementary material Fig. S1B). By immunofluorescence staining with the antibodies, centlein was detected as punctate signals scattered in the cytoplasm and colocalized with the centrosomal marker γ-tubulin (supplementary material Fig. S1C). Silencing centlein reduced the signals from immunofluorescence to below 20% of the negative control (supplementary material Fig. S1C), confirming the specificity of the staining patterns of the antibodies.

To localize centlein at the centrosome precisely, we chose to use RPE-1 cells. Upon serum withdrawal, RPE-1 cells become quiescent and develop primary cilia. At this stage, an RPE-1 cell possesses a basal body/mother centriole and a daughter centriole, which are readily visualized by using an antibody against acetylated tubulin (Graser et al., 2007b) – a marker of primary cilia and centrioles. Centlein was detected as two dots that colocalized with acetylated tubulin but also localized within the PCM, which was marked by staining for pericentrin and γ-tubulin, and the centriolar satellites, which were marked by PCM-1 (Lopes et al., 2011), indicating that centlein is associated with the two centrioles (Fig. 1A). Further examination revealed that centlein localized distinctly from centrin2 and CP110 (also known as CCP110), which are markers of the distal ends of centrioles (Kim et al., 2011) (Fig. 1B; supplementary material Fig. S1D); by contrast, centlein colocalized substantially with the proximal centriolar markers C-Nap1 and Cep135 (Arquint et al., 2012; Fig. 1B; supplementary material Fig. S1D). In conclusion, the monoclonal antibodies specifically recognize the ∼170 -kDa protein centlein and label the proximal ends of centrioles.

Centlein is localized at the proximal ends of centrioles and required for centrosome cohesion. (A) The quiescent RPE-1 cells were co-stained with the indicated antibodies (green) and an antibody against centlein (red). DAPI was used to stain the nuclei (blue). The insets show enlarged views of centrosomes. (B) RPE-1 cells were co-stained with the indicated antibodies (green) and an antibody against centlein (red). Images of the centrosomes were enlarged. (C) The percentage of centrosome splitting upon depletion of centlein, C-Nap1, rootletin or Cep68 from RPE-1 and U2OS cells by using siRNAs. Centrosomes were counted as split when the distance between two centrioles was >2 µm. **P<0.01 compared with NS, mean±s.d., n>300 per group in three independent experiments. NS, nonspecific. (D–G) U2OS cells were transfected for 72 h with a nonspecific siRNA or siRNA duplexes specific for centlein, C-Nap1, rootletin or Cep68 and were then co-stained with the indicated antibodies (green) and an antibody against γ-tubulin (red). Insets show enlarged images of the centrosomes. (H) Quantification of the normalized fluorescence intensity of centlein, C-Nap1, rootletin and Cep68 at the split centrosomes in the indicated siRNA-treated U2OS cells. **P<0.01 compared with NS, mean±s.d., n>50 per group in three independent experiments. Scale bars: 10 µm (A,D–G); 1 µm (A insets; B); 2 µm (D–G insets).

Centlein was first identified by Nakanishi and colleagues; however, its precise function was unknown (Makino et al., 2008; Sakamoto et al., 2008). In HeLa lysates, western blotting analysis, by using a polyclonal antibody against centlein, has previously detected a single band of 90 kDa (Makino et al., 2008). Because the antibody had been generated against a GST-tagged fragment of centlein (amino acid residues 803–953), it is supposed to detect a band corresponding to the predicted molecular mass of human centlein (162 kDa); however, the specificity of the antibody was not verified (Makino et al., 2008), which explains discrepancies in the observed mobility of the protein.

Centlein is required for centrosome cohesion and recruitment of Cep68 to centrosomes

To explore the function of centlein, we depleted the protein by using siRNAs in RPE-1 and U2OS cells and noticed an increased distance between centrosomes in the centlein-depleted interphase cells. When the distance between the parental centrioles is >2 µm, it is termed ‘centrosome splitting’ (Graser et al., 2007a). Centrosome splitting has been observed previously in cells lacking C-Nap1, rootletin or Cep68, albeit with large variation (Bahe et al., 2005; Graser et al., 2007a). The localization of C-Nap1 to the proximal ends of parental centrioles suggests that it is a centriolar docking protein (Mayor et al., 2000), whereas rootletin and Cep68 decorate fibers emanating from the proximal ends of parental centrioles (Bahe et al., 2005; Graser et al., 2007a). We quantified centrosome splitting in cells lacking centlein, C-Nap1, rootletin or Cep68 in comparison with negative controls (Fig. 1C; supplementary material Fig. S2A). In accordance with previous studies, depletion of either C-Nap1 or rootletin produced the highest splitting ratio (Fig. 1C; Graser et al., 2007a), indicating that C-Nap1 and rootletin are particularly important for the tethering of parental centrioles.

Centlein interacts with C-Nap1 and Cep68. (A) Western blotting of endogenous centlein (top left) after immunoprecipitation with an antibody against C-Nap1. HEK293T cells were transfected with Myc–centlein, immunoprecipitated (IP) with either an antibody against C-Nap1 (bottom left) or Myc (right) and then western blotted (IB) with the indicated antibodies. (B) The lysates of HEK293T cells that coexpressed Flag–Cep68 with Myc–centlein were immunoprecipitated with antibodies against the respective tags. Both proteins were detected in reciprocal reactions. (C) Interaction sites between centlein (top left) and C-Nap1 (top right), as well as between centlein (bottom left) and Cep68 (bottom right). The numbers represent amino acid residues. +, red, interaction; −, black, no interaction (D) U2OS cells were transfected with tagged centlein, C-Nap1 or Cep68 alone (top panel), with tagged centlein and C-Nap1 (middle panel) or centlein and Cep68 (bottom panel), and the distribution of proteins was monitored by immunofluorescence microscopy. Scale bars: 10 µm.

To further establish the association of centlein with both C-Nap1 and Cep68, we overexpressed either centlein with C-Nap1 or centlein with Cep68 in U2OS cells. Note that high-level overexpression of centlein led to the formation of filaments throughout the cytoplasm (Fig. 2D, top left panel), whereas ectopic expression of either C-Nap1 or Cep68 did not produce such structures (Fig. 2D, top panels; Bahe et al., 2005; Graser et al., 2007a). Overexpression of either C-Nap1 or Cep68 resulted in their recruitment to centlein filaments (Fig. 2D, middle and lower panels). These data demonstrate that centlein, indeed, interacts with both C-Nap1 and Cep68.

Centlein complexes with C-Nap1 and Cep68 for centrosome cohesion

The results described above suggest that centlein exists in a complex with both C-Nap1 and Cep68. Therefore, an in vitro direct-binding assay was performed. A GST-tagged fragment of centlein (amino acid residues 89–437), immobilized on sepharose beads, selectively bound both transiently expressed Flag–Cep68 and GFP–C-Nap1 in cell extracts (Fig. 3A). This data, along with a lack of interaction between C-Nap1 and Cep68 (data not shown; Graser et al., 2007a), suggests that C-Nap1 complexes with Cep68 through the independent direct interactions of both proteins with centlein.

Centlein complexes with C-Nap1 and Cep68 for centrosome cohesion. (A) In vitro binding of C-Nap1 and Cep68 with a fragment of centlein (amino acid residues 89–437). Protein extracted from HEK293T cells that coexpressed GFP–C-Nap1 and Flag–Cep68 were incubated with GST, GST–centlein 89–437 aa or GST–centlein 901–1191 aa. After extensive washing, proteins extracted from glutathione–Sepharose-4B beads were analyzed by western blotting with the indicated antibodies. (B,C) U2OS cells transfected with NS or centlein siRNA were co-stained with the indicated antibodies (green) and γ-tubulin (red). The insets show enlarged views of centrosomes. (D,E) U2OS cells were first depleted of centlein and then transfected with the full-length or the various truncated forms of GFP–centlein before being subjected to immunostaining analyses with an antibody against GFP (green) and the indicated antibodies (red). The GFP-tagged centlein – including the full-length protein and fragments corresponding to amino acids 1–898, 1–600 and 151–600 – localized and recruited Cep68 to the centrosomes and also rescued centrosome splitting (indicated by γ-tubulin staining). Insets show enlarged centrosomes. (F) The percentage of centrosomes that split upon expression of the full-length or the various truncated forms of GFP–centlein in U2OS cells depleted of centlein. **P<0.01 compared with GFP, mean±s.d., approximately 100 positively transfected cells were counted in each of the three independent experiments. Scale bars: 10 µm (B–E); 2 µm (B–E insets).

To elucidate an in vivo functional role of the complex, we performed knockdown-rescue experiments. We first depleted endogenous centlein by using siRNAs and observed that the centlein-depleted cells displayed split centrosomes (Fig. 3B,C; Fig. 1C). We then transfected various deletion mutants of centlein into those cells and examined the ability of each construct to localize to the centrosomes and its effect on centrosome splitting. Only the centlein mutants comprising the regions required for interaction with C-Nap1 and Cep68 were able to localize to the centrosomes and rescued centrosome splitting induced by centlein depletion (Fig. 3D–F). Thus, these experiments not only demonstrate that centlein is recruited to the centrosome through its C-Nap1-interaction domain but also substantiate the notion that centlein acts as a molecular link within the C-Nap1–centlein–Cep68 complex to mediate centrosome cohesion.

Centlein and Cep68 are Nek2A substrates

Given that C-Nap1, centlein and Cep68 form a protein complex, we asked whether centlein, like C-Nap1 and Cep68, associated with centrosomes in a cell-cycle-dependent manner (Mayor et al., 2000; Graser et al., 2007a). U2OS cells that had been synchronized for stage-specific populations were examined by using immunofluorescence microscopy. Levels of centlein were relatively high at interphase centrosomes but were reduced on mitotic spindle poles (Fig. 4A,B), which is very similar to the pattern observed for C-Nap1 – a well-characterized substrate of Nek2A (Mayor et al., 2000).

Centlein staining, like Cep68, drops on mitotic spindle poles, and both centlein and Cep68 are novel Nek2A substrates. (A) U2OS cells were synchronized with a double thymidine block and then released for 0 (G1/S), 6 (S), 9 (G2), 12 (M) and 18 h (G1) and co-stained with the indicated antibodies (green) and antibody against centrin2 (red). Cell cycle distribution was monitored by FACS analysis (bottom). Insets show enlarged images of the centrosomes stained for the indicated proteins. (B) Quantification of the normalized fluorescence intensity of centlein, Cep68, C-Nap1 and rootletin at the centrosome in U2OS cells at different stages of the cell cycle. **P<0.01 compared with G1 stage, mean±s.d., n>50 per group in three independent experiments. (C) Nek2A binds to centlein and Cep68. The Myc tag was immunoprecipitated (IP) from lysates of HEK293T cells that had been co-transfected with GFP–Nek2A and Myc–centlein. The immunoprecipitates were then western blotted (WB) with an antibody against GFP antibody (left panel). GFP–Nek2A was immunoprecipitated from transfected HEK293T cells with an antibody against GFP and immunoprecipitates were western blotted with an antibody against centlein (middle). HEK293T cells were co-transfected with GFP–Nek2A and Flag–Cep68, lysates were then immunoprecipitated with an antibody against the Flag tag and western blotted with an antibody against GFP antibody (right panel). (D) HEK293T cells were transfected with either Myc–centlein (left panel) or Flag–Cep68 (right panel) alone or together with wild-type (WT) or catalytically inactive (K37R) GFP–Nek2A. HEK293T lysates treated with (+) or without (−) calf intestinal alkaline phosphatase (CIAP) were subjected to immunoblot analysis with the indicated antibodies. (E) U2OS cells were transfected with either GFP–Nek2A WT or GFP–Nek2A K37R and stained with antibodies against γ-tubulin (top two rows), centlein (middle two rows) and Cep68 (bottom two rows). Insets show enlarged images of centrosomes. Scale bars: 10 µm; 2 µm, insets. (F) In vitro kinase assay using WT or K37R Nek2A and the GST–centlein N-terminal fragment (89–437 aa) or the GST–Cep68 N-terminal fragment (1–282 aa). Substrate phosphorylation was revealed by western blotting with antibodies against phosphorylated serine and phosphorylated threonine residues. The arrows indicate phosphorylated centlein or Cep68. (G) A model for centlein acting as a molecular link between C-Nap1 and Cep68. In interphase, centlein localizes at the proximal ends of centrioles and binds with C-Nap1 and Cep68 to participate in centrosome cohesion. In mitosis, centlein dissociates from centrosomes.

Thus, we sought to determine whether centlein and its binding partner Cep68 are Nek2A substrates. First, we examined whether Nek2A bound to and phosphorylated centlein and Cep68 in vivo. By coexpressing GFP–Nek2A and Myc–centlein in HEK293T cells, we demonstrated that Nek2A specifically co-immunoprecipitated with Myc–centlein (Fig. 4C, left), and, in a reciprocal experiment, the endogenous centlein could be readily detected in the GFP–Nek2A immunoprecipitate (Fig. 4C, middle). Similar to this, an antibody against the Flag tag co-precipitated GFP–Nek2A from lysates prepared from HEK293T cells that had been co-transfected with Flag–Cep68 and GFP–Nek2A (Fig. 4C, right). Furthermore, antibodies against the Myc or Flag tags detected more slowly migrating forms of centlein and Cep68 from HEK293T cells expressing Myc–centlein or Flag–Cep68 together with wild-type (WT), but not catalytically inactive (K37R), GFP–Nek2A (Fig. 4D). Treatment of the HEK293T cell extracts with calf intestinal alkaline phosphatase (CIAP) resulted in loss of these slower migrating centlein and Cep68 bands, confirming that the change in the gel mobility of the proteins was due to protein phosphorylation (Fig. 4D). Overexpression of GFP-Nek2A WT, but not GFP–Nek2A K37R, caused the dissociation of centlein and Cep68 from centrosomes, in turn, leading to centrosome splitting in U2OS cells (Fig. 4E). We then determined whether Nek2A could directly phosphorylate centlein and Cep68 in vitro. Bacterially expressed GST-tagged fragments of centlein (amino acid residues 89–437) and Cep68 (amino acid residues 1–282) were purified as substrates. Nek2A protein was immunoprecipitated from HEK293T cells that had been transfected with either GFP–Nek2A WT or GFP–Nek2A K37R. The in vitro kinase assay showed that both centlein and Cep68 were phosphorylated by active but not inactive Nek2A by using the antibodies specific to the phosphorylated serine and threonine residues (Fig. 4F). We thus conclude that centlein and Cep68 are novel Nek2A substrates.

In summary, centlein, together with Cep68, is a newly identified Nek2A substrate and associated with centrosomes in a cell-cycle-dependent manner (Fig. 4G). Knockdown of centlein displaces Cep68 from centrosomes and, in turn, leads to centrosome splitting. In interphase cells, centlein interacts and forms a protein complex with C-Nap1 and Cep68 at the proximal ends of centrioles (Fig. 4G). Centrosome cohesion is, thus, maintained mainly by C-Nap1–rootletin (Bahe et al., 2005; Mardin and Schiebel, 2012) and supported by C-Nap1–centlein–Cep68 (Fig. 4G).

MATERIALS AND METHODS

Plasmid construction and recombinant proteins

Full-length centlein was obtained from HeLa cDNA and cloned into the vectors pEGFP-C1, pEGFP-N1 and pCMV-Myc. The truncated mutants of centlein were cloned into the vectors pCMV-Myc and pEGFP-C1. Full-length C-Nap1 was a gift from Erich A. Nigg (Bahe et al., 2005) and was subcloned into the p3×Flag-CMV vector. The truncated mutants of C-Nap1, full-length Cep68 (obtained from HeLa cDNA) and truncated mutants of Cep68 were cloned into the p3×Flag-CMV vector. GFP-tagged rootletin was a gift from Erich A. Nigg (Graser et al., 2007a). Wild-type Nek2A was obtained from HeLa cDNA and cloned into pEGFP-C1. The catalytically inactive (K37R) Nek2A was gifted from Erich A. Nigg (Bahe et al., 2005). Sequencing was performed for all constructs. For expression of recombinant proteins, base pairs 265–1311 and 2701–3573 of centlein or base pairs 1–846 of Cep68 were amplified and inserted into the expression vector pGEX-4T-1. GST-tagged fragments were expressed in Escherichia coli strain BL21 (DE3) and purified with glutathione–Sepharose-4B (17-0757-01, GE Healthcare Life Sciences).

siRNA experiments

For RNA-mediated interference, cells were transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol and harvested at 72 h post transfection. ON-TARGETplus SMARTpool siRNAs against centlein were obtained from Dharmacon (L-020720-02) targeting the following sequences: 5′-GAGCTGAAGTACACGCAA-3′, 5′-GTTGAAGTATCACAGAGTA-3′, 5′-GCTAGAGGGCATCTCGGTA-3′ and 5′-ACGGAAAATTGCAGT-3′. Several proteins were depleted using siRNA duplex oligonucleotides targeting the following sequences: C-Nap1, 5′-CTGGAAGAGCGTCTAACTGAT-3′ (Bahe et al., 2005); Cep68, 5′-ACCGAAGATGATCCATCCCTA-3′ (Graser et al., 2007a) and rootletin, 5′-AAGCCAGTCTAGACAAGGA-3′ (Bahe et al., 2005). Control siRNAs were also used (ON-TARGETplus Non-Targeting pool, D-001810-10, Dharmacon). Rescue experiments were performed by transfecting siRNA-treated cells 72 h later with the indicated DNA constructs using Lipofectamine 2000 (Invitrogen).

Immunoprecipitation

HEK293T cells cultured in 10-cm dishes were lysed with 1 ml of cold ELB buffer [50 mM HEPES, 250 mM NaCl, 0.1% NP-40, 1 mM PMSF (Sigma) and complete EDTA-free protease inhibitor cocktail (Roche)]. The lysates were centrifuged at 12,000 g for 10 min and the supernatant was precleared by incubation with 50 µl of protein-G–Sepharose (CW0012A, Cowin Biotech). After the mixtures had been incubated at 4°C for 3 h, the supernatant was incubated with 2 µg antibody (homogeneous normal IgG as control) at 4°C for an additional 3 h, followed by addition of 20 µl Dynabeads–protein-G (10004D, Invitrogen) and incubated at 4°C overnight. Beads were washed four times using ELB buffer, followed by heating of the precipitated material in SDS-PAGE sample buffer and immunoblotting analysis.

In vitro binding assays

GST and GST fusion proteins were expressed in E. coli strain BL21 (DE3) and purified by affinity chromatography with glutathione–Sepharose-4B and then crosslinked to Sepharose beads. GST or GST fusion proteins bound to the beads (20 µg) was incubated in ELB buffer with 1 mg of extract of HEK293T cells that had been transiently transfected with GFP–C-Nap1 and Flag–Cep68. The mixture was incubated at 4°C overnight with agitation. After four washes with ELB buffer, proteins were extracted from the Sepharose beads by boiling in SDS-PAGE sample buffer and were then analysed by western blotting (Rountree et al., 2000).

Cell cycle synchronization and FACS analysis

U2OS cells were synchronized with a double thymidine block and then released for 0, 6, 9, 12 and 18 h. The cells were trypsinized, suspended in 100 µl PBS and fixed by adding 3 ml of cold 70% ethanol and incubating overnight. The fixed cells were washed with PBS and incubated with 10 mg/ml propidium iodide (PI) for 1 h. The cells were then analyzed using an Accuri C6 flow cytometer (BD Biosciences).

Measurements and statistical analysis

ImageJ software was used to measure the fluorescence intensity and centrosomal distance. The detailed methods of measurements are detailed elsewhere (Mardin et al., 2010). The statistical significance of the difference between two means was determined using a two-tailed Student's t-test. Differences were considered significant when P<0.01.

Acknowledgments

We thank Erich A. Nigg and Elena Nigg (University of Basel, Switzerland) for kindly providing antibodies against rootletin and Cep68, and plasmids of GFP-C-Nap1, GFP-rootletin and GFP-Nek2A K37R. We are very grateful to Kunsoo Rhee for the CP110 antibody. We also thank the members of Zhihai Qin (Institute of Biophysics, Chinese Academy of Sciences, Beijing) for technical assistance.

Footnotes

G.F., D.Z., H.Y. and L.Z. performed experiments. L.Y., X.B. conceived the idea and planned the research. L.Y. and G.F. wrote the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 31071182, 31271480 to L.Y. and X.B.]; University of Chinese Academy of Sciences [grant numbers 095102GN00, Y15102GN00 to L.Y.]; the National Basic Research Program of China, 973 Program [grant number 2010CB934004 to X.B.].

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